Phosphorylation of eNOS on serine 1179 by Akt is important for eNOS activation in response to agonists such as vascular endothelial growth factor (VEGF), estrogen or flow. In this competitive renewal, we provide data showing that the genetic loss of eNOS severely impairs ischemia-induced arteriogenesis in mice, a phenotype rescued by adenoviral transduction of the ischemic limb with S1179D eNOS, a constitutively active form of eNOS, using a new, more efficient approach for in vivo gene transfer. More importantly, we have observed an identical phenotype of critical limb ischemia in mice deficient in the gene for Akt1, but not for Akt2, suggesting that the Akt-eNOS axis may be critical for arteriogenesis following tissue ischemia. In addition, utilizing caveolin-1 (-/-) mice and new strategies aimed at the hsp90/Akt interaction (dominant negative hsp90 and cell permeable peptides that antagonize the docking of Akt/eNOS to hsp90), we will dissect the importance of caveolin-1 and hsp90 in regulating Akt-dependent eNOS phosphorylation. We hypothesize that a major physiological role of Akt in the cardiovascular system is to modulate eNOS, thus preserve blood flow and promote arterial remodeling and arteriogenesis. As a corollary to this hypothesis, we predict that the scaffolding function of hsp90 is crucial to coupling signal transduction via Akt to eNOS activation. We will: 1. determine the importance of eNOS vs. Akt in a model of ischemia-induced arteriogenesis; 2. dissect the importance of Akt1/Akt 2 in mediating angiogenic phenotypes in vitro; and 3. examine the molecular regulation of the eNOS phosphorylation (via the hsp90/Akt signaling module) in blood vessels, endothelial cells isolated from mice deficient in the negative regulator of eNOS, caveolin-1. We will also manipulate the levels and activity of hsp90 in endothelial cells using new methodologies (dominant negative mutations and cell permeable peptides) to assess the importance of hsp90 in regulating the functions of Akt and eNOS. Collectively, this work will facilitate our understanding of the importance of Akt/eNOS axis in arteriogenesis and vascular remodeling and provide insights into the molecular machinery required for eNOS regulation by protein phosphorylation using a multi-disciplinary approach.